The present invention relates to a method for rapid detection of a Respiratory Syncytial virus (RS virus) related biological cell and/or biological particle contained in a body fluid sample. The method is used for rapidly diagnosing a condition in an individual resulting from an infection by a RS virus.
The method comprises the further steps of detecting a plurality of infection and/or inflammatory response agents, preferably cytokines, and performing a profile of such agents. The profile is a further indication of the condition being diagnosed. The method for detecting a plurality of infection response agents, preferably cytokines, includes the step of performing a profile of such agents.
The methods of the invention are performed by contacting i) a body fluid sample potentially comprising the infectious agent and/or an infection and/or inflammatory response agent, including cytokines, with ii) a targeting species that is preferably quantifiably detectable and capable of specifically recognising a predetermined infectious agent and/or a predetermined infection response agent. It is preferred that the contacting takes place essentially without pretreatment of the body fluid sample.
The targeting species preferably comprises an antibody capable of contacting one or both of an infectious agent and an infection response agent. The targeting species may also comprise a visible label capable of being detected when the complex of the targeting species with either one or both of an infectious agent and an infection response agents is in contact with a solid test area on e.g. a lateral flow device.
Antigens from microbial cells have been detected in the prior art. U.S. Pat. No. 4,663,277 relates to a method for detecting a virus by means of an immunoassay in which an extended solid phase coated with antiviral antibody is employed to bind and remove virions from a specimen by forming an immuno-complex with antigens of said virions, a mobile solid phase comprising a dispersion of microspheres coated with the antiviral antibody is used to bind said microspheres to antigens associated with said Immuno-complex, and the presence of bound microspheres is detected. The detection sensitivity is amplified by using microspheres comprising a dye or a label. The extended solid phase may be in the form of a dipstick, syringe, tube or container that can be easily contacted with the specimen. A virus detection kit provides the extended solid phase and mobile solid phases, each coated with antiviral antibodies.
In one embodiment the invention disclosed in U.S. Pat. No. 4,663,277 pertains to a method for detection of viruses in a specimen and comprises the steps of i) treating the specimen to remove undesired components ii) contacting the specimen with a solid phase support having conjugated thereto antiviral antibody capable of forming immuno-complexes with antigens characteristic of the viruses to be detected, iii) separating the solid phase support from the specimen, iv) contacting the separated solid phase support with a mobile solid phase consisting of dispersed microspheres smaller than 0.1 xcexcm and labelled with metal elements and having conjugated thereto the antiviral antibody that enables the binding of said microspheres to said immuno-complexes, v) separating the unbound mobile solid phase from the solid phase support, and vi) measuring the presence of microspheres bound to said solid phase support by X-ray fluorescence, thereby detecting or determining the presence of viruses in said specimen.
An antispecies antibody is covalently bound to the solid phase support as well as to the mobile solid phase, and an antiviral antibody that forms an immuno-complex with the antispecies antibody is coupled therewith, whereby an antiviral antibody capable of forming immuno-complexes with antigens of viruses to be detected is conjugated to said solid phase support and to said mobile solid phase.
In one embodiment of the invention a plurality of different antiviral antibodies capable of forming complexes with corresponding antigens of different types of viruses are conjugated to the solid phase support as well as to the mobile solid phase, whereby the presence of one or more of a plurality of different types of viruses in the specimen can be detected at the same time.
U.S. Pat. No. 4,740,467 discloses a method for diagnosing syphilis and other treponematoses infections such as yaws and pinta. The method involves admixing i) a biological sample, such as lesion exudate, cerebrospinal fluid, serum, urine, amniofic fluid, synovial fluid or tissue homogenate from a person suspected of having syphilis, yaws or pinta together with ii) a reagent of monoclonal antibodies which are specific for antigens of virulent subspecies of Treponema pallidum, including pertenue, endemicum, carateum and pallidum. If Treponema pallidum, the causative organism of syphilis, is present, an immunological specific binding reaction will occur between the monoclonal antibodies and antigenic sites on T. pallidum cells. A positive immunoreaction is detected directly by a variety of techniques including radioimmunoassay, fluorescent immunoassay, enzyme linked immunosorbent assay, agglutination reactions, and complement consumption tests.
U.S. Pat. No. 5,290,677 discloses a method for detecting hepatitis A virus by capturing whole virus particles with antibodies specific to hepatitis A virus. In subsequent steps the method comprises generating a cDNA copy of the RNA by reverse transcription in the presence of a primer having a predetermined sequence, amplifying the cDNA by a polymerase chain reaction, and detecting the amplified cDNA by hybridization with probes of a predetermined sequence, or by detection of label bound to the primer, wherein the presence of detectable hybridization or amplification indicates the presence of hepatitis A virus. It is disclosed that samples which contain free virus (for example, stool, environmental samples, or other fomite associated material) may be selectively removed from adventitious material by immunoselection of whole virus using a high titer anti-HAV antibody coated onto a solid phase. The viral RNA is then denatured in the presence of a specific primers, and the viral RNA is reverse transcribed to cDNA using standard methodology.
Further examples of diagnostic methods pertaining to the detection of microbial cells are disclosed e.g. in U.S. Pat. No. 6,077,665 relating to a rapid assay for infection in immunodeficient patients such as neonates or immunocompromised patients (e.g. HIV or transplant patients). The method allows diagnosis at initial evaluation, such that antibiotic treatment and confinement to an intensive care unit can be avoided for uninfected patients. The assay can be used for sepsis diagnosis including the detection of bacterial, viral, or fungal colonization of the blood stream, cerebrospinal fluid (CSF), or urinary tract. The method is based on the measurement of polymorphonuclear leukocyte (PMN, neutrophil) CD11b (Mac-1, CR3) levels by flow cytometry or laser scanning microscopy in whole blood samples.
U.S. Pat. No. 5,965,354 relates to a method and immunodiagnostic test kits for diagnosing herpes simplex virus infection. The methods and kits employ type-specific or type-common antigens in a single-step assay format. In one embodiment the method of the invention comprises the steps of i) contacting a biological sample from a human suspected of containing antibodies to herpes simplex virus with one or more purified herpes simplex virus polypeptides bound to a solid support, under conditions that allow herpes simplex virus antibodies, when present in the biological sample, to bind to said herpes simplex virus polypeptides, and ii) detecting the presence or absence of bound antibodies as an indication of the presence or absence of herpes simplex virus, wherein said detecting is done by using at least one detectably labeled anti-human immunoglobulin antibody.
U.S. Pat. No. 5,939,254 discloses specific primers that amplify a portion of the 3xe2x80x2-noncoding regions of a dengue virus, and a method of using these primers in a rapid reverse transcriptase-polymerase chain reaction (RT-PCR) for specific detection of dengue viruses.
U.S. Pat. No. 5,919,616 relates to serological detection of a herpes simplex virus infection by means of reaction of a patient serum sample potentially containing virus antibody with a virus specific peptide that may be used in an assay including an enzyme linked immunosorbent assay (ELISA).
U.S. Pat. No. 5,744,299 is concerned with a method for evaluating a biological sample for the presence or absence of human parainfluenza virus and for the quantitation of the virus The method comprises the steps of isolating RNA from the biological sample, generating cDNA from the isolated RNA, amplifying the generated cDNA, and determining virus infection by detecting the amplified sequence.
U.S. Pat. No. 5,695,930 relates to a method for detecting antibodies to a human immunodeficiency virus and comprises the steps of i) contacting saliva from a human with p17 protein from human immunodeficiency virus bound to a nitrocellulose-containing solid support for a time and under conditions sufficient for an antibody in the saliva to said antigen to form a complex therewith, and ii) subjecting the complex to detecting means in order to detect the complex.
U.S. Pat. No. 5,660,979 discloses a method for determining virus replication in human cells by human retrovirus using RNA amplification and comprises the step of detecting the hybridization of an RNA probe which specifically hybridizes with spliced RNA and not with genomic RNA. The method permits early detection of RNA replication resulting from primary infection without detecting non-replicating virus.
U.S. Pat. No. 5,643,714 relates to HTLV gp21 envelope protein specific peptides for use in diagnostic assays for detecting and confirming HTLV infection in human sera. The invention also pertains to a kit for detecting the presence of HTLV infection in a human serum sample. The kit comprises i) a solid support and, ii) a peptide antigen attached to the solid support in a reaction zone, and iii) reporter means for detecting the presence of human antibodies bound to the support.
U.S. Pat. No. 5,593,849 discloses an immunochemical assay that uses enzyme-linked immunosorbence to detect the presence of antibodies against environmental protein sequences that mimic the human opioid peptide dynorphin in a human body fluid sample. The assay makes it possible to correlate and diagnose psychobiological or medical disorders related to alterations In the normal levels of dynorphin peptides or their receptors.
U.S. Pat. No. 5,587,285 describes a highly sensitive anti-HIV antibody detection assay. The assay detects the presence of anti-HIV antibodies through the use of a non-denatured HIV antigenic determinant which immunoreactivity binds anti-HIV antibodies in a biological sample. The non-denatured HIV antigenic determinant has provided a means for detecting anti-HIV antibodies in serum samples testing seronegative for the presence of HIV antibodies directed against denatured HIV antigens.
U.S. Pat. No. 5,565,319 relates to compositions derived from a viral isolate of feline T-tymphotropic lentivirus (FTLV) and antibodies to antigenic sites on the virus. The compositions are useful in a variety of techniques for the detection of and vaccination against FTLV. Detection methods disclosed include immunoassays. In one embodiment there is provided an enzyme-linked immunosorbent assay (ELISA) for detecting Feline Immunodeficiency Virus (FIV) antibodies. The assay comprises a solid phase coated with FIV antigen, wherein FIV antibodies in a sample exposed to the solid phase will bind to the antigen; and a detectable label conjugate which will bind to FIV antibodies bound to the solid phase.
U.S. Pat. No. 5,487,969 pertains to a method for detecting the presence of herpes B virus in an individual and comprises the steps of i) obtaining a sample from an individual suspected of being infected with herpes B virus, ii) extracting DNA from any herpes B virus, iii) amplifying segments of the extracted DNA by using predetermined primer sequences, iv) analyzing the amplified DNA segments by means of e.g. digesting the amplified DNA segments with a restriction enzyme, or by hybridizing the amplified DNA segments with a labeled oligonucleotide probe.
U.S. Pat. No. 5,225,322 relates to a method for detecting an antigen in a test sample suspected of containing said antigen, and simultaneously determining a fingerprint of antibodies specific for said antigen. The method comprises the steps of i) providing polyclonal antibodies specific for the antigen in question, ii) separating the polyclonal antibodies from each other according to the electrical charge of individual antibodies, iii) binding the separated antibodies to a solid support so that the antibodies separated in step ii) maintain the same relative position with respect to each other on the solid support, the relative position of the antibodies forming a fingerprint of antibodies specific for said antigen, iv) contacting the antibodies bound in step iii) with a test sample suspected of containing the antigen under conditions selected to allow binding of the antigen to the antibodies bound in step iii), v) contacting antigen bound in step iv) with detectably labeled antibodies specific for the antigen, under conditions selected to allow binding of said detectably labeled antibodies to said antigen, and vi) detecting the detectably labeled antibody as an indication of the presence of said antigen in the test sample, and revealing the fingerprint of antibodies that are specific for the antigen when the antigen is present in the test sample.
U.S. Pat. No. 5,212,062 is related to a method for detecting antigens from Chlamydia psittaci or Chiamydia trachomatis in a sample. The method comprises the steps of i) contacting a sample with a predetermined monoclonal antibody affixed to a solid support for a time and under conditions sufficient to form an immune complex on said support, ii) contacting the support with an antibody which binds to said antigen in said immune complex for a time and under conditions sufficient for binding to occur, and iii) detecting the presence of said immune complex as an indication of the presence of Chlamydia psittaci or Chiamydia trachomatis antigen in said sample.
U.S. Pat. No. 5,155,021 is directed to a method for the determination of a herpes simplex virus and comprises a first step i) of contacting a specimen suspected of containing herpes simplex viral antigen with polymeric particles which have a surface area of from about 0.1 to about 600 m2/g of particles. Each particle is substantially free of any chemical or biological material and has an average diameter of from about 0.01 to about 10 xcexcm. The particles are capable of directly binding herpes simplex viral antigen. Within about 10 minutes of contacting step i) A, herpes simplex viral antigen directly bound to the particles are contacted in a second step ii) with herpes simplex viral antibody so as to form an immunological complex on said particles. The bound complex is then separated from uncomplexed herpes simplex viral antibody by using a microporous membrane having an average pore size of from about 0.1 to about 20 xcexcm. and the complex is determined as an indication of the presence of herpes simplex virus in said specimen. The method is carried out within about 30 minutes.
U.S. Pat. No. 5,093,230 relates to an assay method for detecting 1xcexcM antibodies to a retrovirus selected from the group consisting of HIV-1, HIV-2, HTLV-I, and HTLV-II. The method is carried out within 70 minutes and comprises the steps of i) contacting nitrocellulose paper containing blotted, resolved retrovirus antigen protein obtained from gel electrophoroctically received viral lysate with a test sample, and incubating under predetermined conditions the nitrocellulose paper and test sample to permit binding of antibodies present in the sample to the protein on the nitrocellulose paper, iii) contacting the incubated nitrocellulose paper of step i) with an anti-IgM enzyme conjugated antiserum reactive with said antibodies, and incubating to permit binding of the antiserum to said antibodies, iii) contacting the incubated nitrocellulose paper of step ii) with an enzyme substrate specific for the enzyme of step ii), and incubating to produce a colour, iv) stopping the colour producing reaction of step iii); and v) evaluating the amount of colour produced as an indication of the presence of antibodies to the viral lysate.
A plurality of cytokines have been detected in the prior art. U.S. Pat. No. 5,587,294 relates to a method for measuring endogenous cytokines in blood. Cytokines are measured in the presence of substances that bind the cytokines and causes conventional methods to give inaccurate results. The invention also pertains to non-invasive measurement of cytokines in biological fluids such as saliva and nasal secretions. In one embodiment the invention relates to a method for monitoring cytokine therapy in a human, wherein the cytokine is able to bind a carrier molecule, with the proviso that the cytokine is not IL-1. The method comprises the steps of i) obtaining a human body fluid sample potentially comprising a cytokine, ii) forming an assay mixture by combining the sample from step i) with a) an antibody capable of binding specifically to substantially all of the cytokine, wherein the antibody is immobilized on a solid phase support, and b) a labeled binding epitope of the cytokine, wherein the labeled binding epitope competes with the cytokine for antibody binding, iii) incubating the assay mixture to allow the immobilized antibody to bind specifically to either the cytokine or the labeled binding epitope, iv) washing unbound, labeled binding epitope from the solid phase support, v) detecting bound label on the solid phase support, vi) determining the amount of the cytokine in the sample. In a further step the determination of the amount of cytokine is compared to the amount of the cytokine in the sample with a determination of the cytokine in a previous body fluid sample. Preferred cytokines are interleukin-2, interleukin-6, interferon-xcex1, interferon-xcex3 and tumor necrosis factor-xcex1.
In a first aspect there is provided a kit for directly detecting a predetermined RS virus related biological cell present in a sample in an amount of less than about 2000 per microliter (10xe2x88x926 liter), said kit comprising
i) a solid support and
ii) a plurality of a first targeting species bound to the solid support, said targeting species being capable of directly detecting said predetermined RS virus related biological cell when it is present in a sample that is brought into contact with the solid support, and
iii) a conjugate comprising a polymeric carrier molecule bound to at least
a) one first and/or second targeting species being capable of directly detecting said predetermined RS virus related biological cell when it is present in a sample that is brought into contact with the solid support, and
b) at least one labelling species.
In further aspects the invention relates to a method for detecting a RS virus related biological cell in a sample and a method for diagnosing and/or treating an infection in an individual.
The method of detecting a predetermined RS virus related biological cell present in a sample, preferably a body fluid sample, comprises the steps of
i) contacting the sample with the kit according to the invention, and
ii) detecting a targeting species capable of targeting the predetermined RS virus related biological cell,
wherein the detection of the targeting species is indicative of the presence of the RS virus related biological cell in the sample.
In another aspect there is provided a method for detecting at least one predetermined inflammatory indicator present in a sample in an amount of less than 100 nanograms (100xc3x9710xe2x88x929 grams) per milliliter (10xe2x88x923 liter). The method comprises the steps of
i) contacting the sample with a kit comprising
a) a solid support, and
b) a plurality of a first targeting species bound to the solid support, said targeting species being capable of directly detecting said predetermined RS virus related biological cell when it is present in a sample that is brought into contact with the solid support, and
c) a conjugate comprising a polymeric carrier molecule bound to i) at least one first and/or second targeting species capable of directly detecting said predetermined RS virus related biological cell when it is present in a sample that is brought into contact with the solid support, and ii) at least one labelling species, and
ii) detecting a targeting species capable of targeting the predetermined inflammatory indicator,
wherein the detection of the targeting species is indicative of the presence of the predetermined inflammatory indicator in the sample.
By the term inflammatory indicator or agent is meant an indicator of inflammatory and/or immune system activity, such indicators for example being cytokines and autoantibodies.
In a further aspect there is provided a method for diagnosing a RS virus infectious condition in an individual, said method comprising the steps of
i) detecting a predetermined RS virus related biological cell present in a body fluid sample according to the method of the invention, and
ii) diagnosing said infectious condition.
In a still further aspect the invention pertains to a method for diagnosing a RS virus infectious condition in an individual, said method comprising the steps of
i) detecting a predetermined RS virus related biological cell present in a body fluid sample according to a method of the invention,
ii) detecting a predetermined inflammatory indicator present in a body fluid sample according to a method of the invention, and
iii) diagnosing said infectious condition.
In yet another aspect there is provided a method for treating a RS virus infectious condition in an individual, said method comprising the steps of
i) performing a diagnosis according to any of the methods of the invention, and
ii) treating the RS virus infectious condition based on the diagnosis.
Still further aspects of the invention relates to a kit according to the invention for use in i) a method for detecting a predetermined RS virus related biological cell or a predetermined inflammatory indicator, ii) a method for diagnosing a RS virus infectious condition in an individual, and/or a method for treating a RS virus infectious condition in an individual.